Rapid and effective healing of burn wounds with cultured analogs of human skin is the central objective of this proposal. Medical benefits from improved healing may include, but not be limited to; reduced requirements for split- thickness autograft, shorter hospitalization time; and reduced long-term morbidity after recovery. However, anatomic and physiologic deficiencies of all current models of cultured skin have restricted realization of these benefits. Major deficiencies result from culture conditions that do not generate an epidermal analog with fully functional barrier properties, from irregular pigmentation after healing, and from absence of a vascular plexus in the dermal analog which delays vascularization. The current model of cultured skin substitute (CSS) in this laboratory is a collagen- based sponge populated with cultured human keratinocytes and fibroblasts. An investigative cycle has been established to: A) design experiments to address clinical deficiencies of CSS, B) perform mechanistic studies in vitro to generate new prototypes of CSS; C) transplant experimental CSS to athymic mice to measure efficacy and tissue phenotypes, and, D) study the improved prototype of CSS for reduction of mortality and morbidity in critically injured burn patients. Six specific aims will be pursued: 1 ) Regulation of cellular viability (DNA synthesis, mitochondrial metabolism) and phenotypes (epidermal barrier, basement membrane) by culture conditions (media, biophysical environment); 2) Identification of molecular mediators (cytokines, extracellular matrix) of wound healing processes (angiogenesis, matrix structure); 3) Regulation of melanocyte distribution (cell density) and pigment expression (melanin content) to restore normal skin color; 4) Stimulation of angiogenesis by addition of human dermal microvascular endothelial cells and morphogenesis of vascular analogs; 5) Regulation and automation of keratinocyte growth rates and metabolism (reduction of lactic acid and ammonia) in the Kerator bioreactor; and, 6) Treatment of extensive burns with cultured skin substitutes in the clinic by paired-site comparison to meshed, split-thickness skin autograft. Comparative parameters will include: a) quantitative wound closure with skin substitutes (rates of engraftment, ratio of healed area to biopsy area, frequency of regraftng); and, b) qualitative outcome (scarring, contraction, function and cosmesis). The investigators possess all of the required expertise in cell biology, skin biochemistry and biophysics, wound physiology, chemical engineering and clinical burn care to perform these studies successfully. Accomplishment of these objectives will contribute to reduced mortality and morbidity from burns, improved materials for plastic and reconstructive surgery, and development of other tissue and organ substitutes.